Treatment of Melanoma Using HSV Mutant

ABSTRACT

Use as an anti-cancer agent of a mutant herpes simplex virus wherein the mutant virus comprises a modification in the γ34.5 gene in the long repeat region (R L ) such that the γ34.5 gene is a non-functional, manufacture of medicaments and methods of testing cancer in mammals employing HSV mutant.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation in part of pending U.S. patentapplication Ser. No. 12/692,347, filed Jan. 22, 2010, which is acontinuation of U.S. patent application Ser. No. 11/152,591, filed Jun.13, 2005 (issued U.S. Pat. No. 7,674,468), which is a continuation ofU.S. patent application Ser. No. 08/776,350, filed Apr. 18, 1997, nowabandoned, which is the U.S. National Stage of International ApplicationNo. PCT/GB95/01791 filed Jul. 28, 1995 (published in English under PCTArticle 21(2)), which in turn claims the benefit of Great BritainApplication No. 9415320.2, filed Jul. 29, 1994. These applications areincorporated by reference herein their entirety; this application isalso a continuation in part of pending U.S. patent application Ser. No.13/090,983, filed Apr. 20, 2011, which is a continuation of Ser. No.11/765,189, filed Jun. 19, 2007, now abandoned, which is a continuationof U.S. application Ser. No. 11/148,575, filed Jun. 8, 2005, nowabandoned, which is a continuation of U.S. application Ser. No.09/117,218, filed Jan. 11, 1999, now abandoned, which is the U.S.National Stage of International Application No. PCT/GB97/00232, filedJan. 27, 1997 (published in English under PCT Article 21(2)), which inturn claims the benefit of Great Britain Application No. 9601507.8,filed Jan. 25, 1996, and Great Britain Application No. 9623365.5, filedNov. 9, 1996. These applications are incorporated by reference hereintheir entirety.

FIELD OF INVENTION

The present invention relates to the use of a herpes simplex virus (HSV)mutant for the treatment of cancer tumors, particularly those of thebrain or nervous system whether the tumors are metastatic tumors orprimary tumors.

BACKGROUND

The DNA sequence of herpes simplex type 1 HSV-1) is known (references13,25) and is linear with a length of about 152 k residues. It consistsof two covalently linked segments, designated long (L) and short (S).Each segment contains a unique sequence flanked by a pair of invertedterminal repeat sequences. The long repeat (R_(L)) and short repeat(R_(S)) are distinct. The unique long (U_(L)) region includes genes UL1to UL56, and the U_(S) region includes genes US1 to US12.

A relatively large number of patients with advanced cancers will developmetastatic lesions in the brain and spinal cord. This frequently resultsin severe and debilitating neurological complications includingheadache, paralysis, seizures, and impaired cognition. It has beenestimated that 70,000 cancer deaths occur each year in the United Stateswith metastatic lesions to the central nervous system (CNS). Radiationand steroids are presently the principle therapies used, however, theyare only palliative, and frequently cause significant neuropsychologicaland endocrinological morbidity. Surgery is generally reserved forremoval of solitary metastases, and is often not curative (1).

Viral therapy for the destruction of tumors is not a new concept.Effects in various experimental tumor systems have been demonstratedusing parvovirus H-1, Newcastle disease virus, retroviral vectorscontaining drug susceptibility genes, and Herpes Simplex Type I virus(HSV-1) (2-7). The mechanisms by which viruses improve the outcome inexperimental tumor systems are complex and poorly understood. Braintumors represent a dividing cell population occurring within anessentially non-dividing cell population of support cells, andterminally differentiated neurons. Thus, in the context of brain tumortherapy, one rationale is to select a virus that replicates exclusivelyor preferentially in dividing cells. Such a virus may be capable ofestablishing a lytic infection exclusively in tumor cells within theCNS, ultimately destroying the tumors without infecting surroundingbrain, and without deleterious effects to the host.

Pioneering experiments with HSV showed a dose dependent improvement insurvival of nude mice bearing intracranial human gliomas followingintratumoral therapy with mutant HSV-1 dlspTK (3). This virus has adeletion in the viral thymidine kinase (TK) gene, (8) and exhibits arelatively neuro-attenuated phenotype in mice (9). However, dlspTKinfection of tumor bearing animals causes histologically evidentencephalitis (3). The use of TK⁻ mutants of HSV-1 for viral therapy alsohas an inherent major disadvantage in that these viruses are resistantto the clinically effective anti-viral agents acyclovir and ganciclovir(10).

The terminal 1 kb of the long repeat region (R_(L)) of the HSV-1 andHSV-2 genomes contain a gene (11-13), that confers neurovirulence.Deletion or mutation of this gene (γ34.5), results in variants that growas well as wild type virus on dividing cells of many established celllines, but show impaired replication on non-dividing cells (12-14). Inmice, γ34.5 null mutants are incapable of replicating in the centralnervous system, and do not cause encephalitis (12, 15-16).

A mutant HSV-1 called R3616, containing a 1000 base pair (bp) deletionin γ34.5, with an LD₅₀ (minimum dose of virus that kills 50% of infectedanimals) that is at least 3×10³ fold greater than wild type F strainvirus from which it was derived (12), has been shown to improve theoutcome of nude mice bearing intracranial human gliomas (17). In thework presented here, we have utilized an HSV-1 strain 17 mutant viruscalled 1716, that has a 759 bp deletion in γ34.5 (16).

The construction of mutant virus 1716 is described in published patentapplication WO92/13943 (PCT/GB92/00179) the contents of which areincorporated herein by reference. However, this patent publication issolely concerned with the use of mutant 1716 as a vaccine, either initself or as a vector vaccine which includes a heterologous gene codingfor an antigen.

Melanoma is a prevalent malignancy. Cerebral metastases occur in up to75% of patients with metastatic disease, and are among the most commoncauses of death (18-22). Presently, the life span of patients with CNSmelanoma is short, ranging from 2 to 7 months (23).

It is an object of the present invention to provide an improvedHSV-based viral therapy of cancer tumors.

STATEMENT OF INVENTION

The present invention in one aspect provides the use as an anticanceragent of a mutant herpes simplex virus which has been modified in theγ34.5 gene of the long repeat region (R_(L)) such that the gene isnon-functional.

The invention also relates to a method of treatment of cancer in amammal (human or animal) by the administration to the mammal of ananti-cancer effective dose of the mutant herpes simplex virus.

DETAILED DESCRIPTION

For the purposes of the present invention “non-functional” means thatthe gene has been modified by deletion, insertion or substitution (orother change in the DNA sequence such as by rearrangement) such that itdoes not express the normal product or a functionally equivalentproduct. The effect of the non-functionality of the gene is that theneurovirulence of the virus to the patient is substantially removed.

Thus the invention is based on the finding that rendering the γ34.5 genenon-functional provides an HSV mutant which is particularly effective indestroying dividing tumor cells, whilst at the same time the HSV mutantdoes not replicate within normal non-cancerous cells. It therefore hasthe potential to provide a safe anti-cancer treatment.

Two types of herpes simplex virus are known HSV-1 and HSV-2 and eithermay be employed in the present invention to provide the HSV mutant.Inter-type recombinants containing DNA from both types could also beused.

The modification may be effected at any convenient point within theγ34.5 gene, and such point generally corresponds to a restriction enzymesite. The modification may be within the Bam H1 a restriction fragmentof the R_(L) terminal repeat (corresponding to 0-0.02 and 0.81-0.83 mu).The modification is typically a deletion of 0.1 to 3 kb, particularly0.7 to 2.5 kb. In this work a 759 bp deletion in γ34.5 was made in theHSV-1 mutant, referred to as 1716.

The HSV genome also includes a number of other genes which arenon-essential to the successful culturing of the virus. It is, ofcourse, necessary to retain within the HSV mutant the ability to culturethe mutant so that the mutant is self-replicating and stocks of themutant can be grown in tissue culture. Lethal modifications of thegenome which remove the ability to culture the HSV mutant are notacceptable.

However, in addition to the primary modification to the γ34.5 gene ofthe R_(L) region, it may be advantageous to also include in the HSVmutant one or more secondary non-lethal modifications withinnon-essential genes.

The present invention also encompasses as a new product an HSV mutantwhich includes in addition to the primary modification, a secondarynon-lethal modification (for example within Vmw65). The mutant may bederived from HSV-1 or HSV-2.

In a similar way, other secondary modifications may involve modificationof the latency associated transcript (LAT) promoter so as to render thepromoter non-functional and prevent transcription thereof.

Herpes simplex virus infects the brain and nervous system. The HSVmutant is effective against primary tumors originating within the brainand nervous system, but is particularly useful against metastatic tumorswhere cancer cells originating elsewhere have lodged in the brain ornervous system (particularly the central nervous system (CNS)). Brainmetastases occur commonly in a variety of human cancers (e.g.melanomas), and at present such cases are invariably fatal. The efficacyof treatment according to the invention employing the HSV mutant willdepend on the time after origination of the tumor at which the treatmentis initiated, but efficacy is improved by early treatment for example in1 to 30 days.

The LD₅₀ (minimum dose of virus that kills 50% of infected animals) ofthe 1716 mutant in respect of mice is 10⁶ fold greater than that of thewild type 17+ virus from which it is derived. Thus the neurovirulence of1716 is essentially removed relative to the wild type virus.

The effective non-toxic dose of HSV mutant can be determined by routineinvestigation by the skilled addressee, and will depend on a number offactors including the particular species of mammal and the extent ofdevelopment of the tumor. A guide can be obtained from the Examplesherein.

In a further aspect of the invention there is provided a method oftreating cancer in mammals, in particular in humans by administering apharmaceutical formulation comprising the HSV mutant to mammals, inparticular to humans. Thus, the method of treatment can comprise theadministration of a pharmaceutical formulation comprising the HSV mutantby injection directly into the tumour or parenterally into the bloodstream feeding the tumour.

It will usually be presented as a pharmaceutical formulation including acarrier or excipient, for example an injectable carrier such as salineor apyrogenic water. The formulation may be prepared by conventionalmeans.

Embodiments of the invention will now described by way of example only.

FIGS. 1 and 2 show the results of experiments described fully inExamples 3 and 5 respectively.

FIG. 1: Survival Curves

Tumor-bearing mice injected at 10 days (FIG. 1 b) and at 5 days (FIG. 1b) post tumor injection with HSV-1 mutant 1716.

FIG. 2: Relative replication rates of HSV-1 mutant

Relative replication rates of HSV-1 mutant 1716 in brain tumor (closedsquares); and of 1716 and wild type 17+ in non-tumor brain (open squaresand closed triangles respectively).

FIG. 3: HSV-1 Genome map.

HSV-1 genome showing approximate location of γ34.5, 2 kb latencyassociated transcript (LAT) and neighbouring genes. A. The 152 kb HSV-1strain 17+ genome, illustrating the unique long and short segments ofthe genome, U_(L) and U_(S) (lines), bounded by internal (IR) andterminal repeat (TR) regions (open boxes). Hatch marks show location ofthe VP16, thymidine kinase (TK) and glycoprotein C (gC) genes. B.Expanded view of the U_(L)/U_(S) region of the genome the location ofthe γ34.5, ICPO and ICP4 mRNAs and the location of the 2.0 kb LAT whichis expressed during acute and latent infection. C. The location of the759 bp deletion in strain 1716. D. The location of the LAT specificBstEII-BstEII probe used for in situ detection of HSV specific geneexpression. Nucleotide positions are based on DNA sequence analysis ofPerry and McGeoch (45).

FIG. 4: Quantification of infectious virus in nude mouse brain after ICinoculation.

To investigate the extent of strain 1716 replication in brain tumors,nude mice were injected with NT2 cells. Twelve days later each mouse wasinfected with 5×10⁵ PFU of strain 1716 at the same stereotacticcoordinates (open circles). At the times indicated, mice weresacrificed, the brains were frozen in Liquid N₂ and stored at −70° C.Specimens were thawed rapidly, homogenized, and viral titration wasperformed in triplicate on BHK cells. To establish the growthcharacteristic of strain 1716 and parental 17⁺ in brain without tumor,mice were injected intracranially with either 5×10⁴ PFU of 1716 (closedcircles) or 1×10⁶ PFU of 17⁺ (closed triangles). Mice were sacrificed atthe times shown and processed as described in Methods. Each point is themean of 2 mice with SEM bars.

FIG. 5: Detection of replicating virus by immunohistochemistry and insitu hybridization

Nude mice were IC injected with 3×10⁴ NT2 cells. 14 days later they wereinoculated with 5×10 PFU of 1716. A. Control mice with tumor after 14days. B. Tumor is histologically very diverse, arrows: tubularstructures. C. MOC-1 antibody specifically identifies the NT2 tumorcells. D. Antibody MIB-1 identifies cycling cells. Note low number oflabeled cells in tubular structures (asterisk). E. 14 day tumor afterthree days infection with strain 1716. The arrow indicates a region ofextensive tumor lysis and necrosis. F. Infected cells showcharacteristic features of herpes infected cells such as intranuclearinclusion bodies formation, cytomegaly and necrosis. G. Herpes antigenis limited to tumor cells. H. Virus replicates in tumor cells atinterface between tumor and host brain shown using anti-HSV antibodies.I. The kinetics of viral replication are delayed at 3 days afterinfection in the tubular structures compared to surrounding non-tubularcells as shown with anti-HSV antibodies. The arrow identifies a rare HSVantigen positive tubular cell. These tubular structures are lysed atlater days after infection. J. Herpes gene expression is also limited totumor by in situ hybridization (black grains). K and L. Wild-type virus(17⁺) replicates in brain and tumor and spreads throughout the wholebrain. M. H&E of 14 day tumor 18 days after infection with strain 1716.Note the small size of tumor. N. Viral antigen and O. viral geneexpression is strikingly limited to the residual tumor mass.Abbreviations: T=tumor; B=host brain

(Scale Bar:=1.2 mm in A; =62.5 μm in B, C, D; =2.0 mm in E&G; =12 μm inF; =450 μm in H, M, N, O; =90 μm in I; 200 μm in J: =900 μm in K and L).

FIG. 6: MRI analysis of treated and untreated NT2 tumors.

Nude mice were injected stereotactically with 3×10⁴ NT2 cells and 11days later (d11 post-tumor cell implantation) T1 weighted,gadolinium-enhanced. MRIs (A, E) were performed. The presence of a tumor(T) is confirmed by the white enhancing lesion appearing in the superiorright hemisphere in these mice. These sections show the area of themaximal tumor mass in cross section. The following day these mice wereinoculated with either 5×10⁵ PFU of strain 1716 or culture medium. Incontrol mice, the tumor progressed over time and IC volume increaseddramatically (B: day 32 post-tumor: C & D: day 41 post-tumor). In strain1716 treated mice the tumor regressed and showed no evidence of livetumor cells or virus in the brain (F & G: day 36 post-tumor), (ScaleBar: =2.77 mm in D & G).

FIG. 7: Electronmicroscopy

Tumors from nude mice which were mock inoculated or inoculated withstrain 1716 were harvested and processed for electronmicroscopy. A:Electronmicrograph of uninfected tumor cells. B: EM of NT2 tumor cellsearly in infection DNA condensation and viral domains (open arrow) canbe observed; C: NT2 tumor cells late in infection, marginated chromatin(*) and viral particles (arrowhead) can be observed, D: A dividing cellthat is infected, arrow: nuclear membrane, c: cytoplasm, n: nucleus(original magnifications at 2500×).

FIG. 8: Prolonged survival of NT2 tumor bearing mice treated with strain1716.

A: Survival Experiment (Table 1, Study V)—20 nude mice werestereotactically injected with 3×10⁴ NT2 cells. Twelve days later, 10mice were sterotactically injected with 5×10⁵ PFU/5 μl of strain 1716(treated, closed circle) and 10 mice were mock injected with 5 μl ofviral culture medium (mock, closed triangle). B: Survival Experiment(Table 1, Study VI)—17 nude mice were stereotactically injected with3×10⁴ NT2 cells. Ten days later, 9 mice were stereotactically injected5×10⁵ PFU/5 μl of strain 1716 (treated, closed circle) and 8 mice weremock injected with 5 μl of viral culture medium (mock, closed triangle).C: Weight Graph—Weights of control (closed triangle) and treated mice(closed circles) from Study VI (Table 1; FIG. 6B). D: Weights of treatedgroup separated into long-term survivors (HR, closed circle) and dead(LR, closed square) compared with strain 1716 alone treated mice (Study1, Table 1; closed triangle). Standard Error of the Mean (SEM) bars areincluded.

FIG. 9: Detection of virus in long-term survivors byimmunohistochemistry and in situ hybridization.

Long-term survivors were sacrificed and brains and other organs werefixed, sectioned and used for immunohistochemical detection of NT2 cellsand HSV and in situ detection of HSV. A. The arrow identifies residualscar at tumor implantation site. B: On histology the brain shows noevidence of any tumor cells (arrow) or replicating virus. C: Theresidual scar site consists of dystrophic calcifications. This is ahigher power view of region identified by the arrow in B. D: Latentvirus was observed in the hippocampus (asterisk) of these survivors (4months post-infection) and the insert shows the nuclear localised signalof LAT positive cells. E: MBP staining shows no evidence ofdemyelination in the whole brains of these mice. F: Dark-fieldphotomicrograph of in situ hybridization performed using a radiolabeledpoly(dT) probe to detect total poly (A)⁺RNA in cells as a measure ofmetabolic health of the LAT positive cells (asterisk). The experimentaltissue (A serial section from 7D) was compared to uninfected, mockinfected and RNAse treated tissue. There was no detectable difference inthe signal in LAT positive area in adjacent serial section. (Scale Bar:1.2 mm in A, B, E; =113 μm in D and −90 μm in insert; =113 μm in F; =4μm in C).

EXAMPLES Section 1 Materials and Methods Animals:

Female C57B1/6 mice (4 to 6 weeks old—weight approximately 20 g) wereobtained from The Jackson Laboratory (Bar Harbor, Me.),

Tumor Cells:

S91 Cloudman melanoma cells were obtained from the ATCC (Rockville,Md.). B16, and Harding-Passey melanoma cells were a generous gift fromDorothee Herlyn (Wistar Institute, Phila, Pa.). Cells were grown inplastic flasks in. AUTO-POW media containing penicillin, streptomycin,and 5% calf serum. When originally obtained, all cell lines were grownup, and then frozen in 95% calf serum/5% DMSO, so that all experimentscould be initiated with cells of a similar passage number. On the day ofintracranial injection, cells in sub-confluent monolayer culture werepassaged with 0.25% trypsin solution in EDTA, washed ×1 in cell culturemedia, resuspended at the appropriate concentration in media withoutserum and held on ice.

Intracranial Tumor Production:

Mice were anaesthetized with I.M. ketamine/xylazine (87 mg/kgketamine/13 mg/kg xylazine). The head was cleansed with 70% EtOH. Asmall midline incision was made in the skin of the head exposing theskull. Stereotactic injection of tumor cell suspension was performedusing a small animal stereotactic apparatus (Kopf Instruments, Tujunga,Calif.). Injections were done with a Hamilton syringe through adisposable 28 g. needle. The needle was positioned at a point 2 mmcaudal of the bregma and 1 mm left of midline. Using a separate 27 g.needle with a shield that limits the length of the needle exposed to 0.5mm, the skull was breached at the appropriate coordinates. The injectionneedle was advanced through the hole in the skull to a depth of 2 mmfrom the skull surface and then backed-out 0.5 mm to create a potentialspace. 1×10⁵ cells in a total volume of 2 μL were injected over 1minute. Following the injection, the needle was left in place for 3minutes, and then slowly withdrawn. The skin was sutured closed.

Virus:

To produce virus stocks, subconfluent monolayers of baby hamster kidney21 clone 13 (BHK) cells were infected with HSV strains in 1814, 1716,dlspTK, or wild type 17+. Virus was concentrated from the culture andtitrated by plaque assay as previously described (28). All viral stockswere stored frozen in viral culture medium (AUTO-POW media containingpenicillin, and streptomycin) at −70° C., and thawed rapidly just priorto use.

Viral Inoculation:

Mice were anesthetized with ketamine/xylazine, and the head was cleansedwith 70% EtOH. Using a Hamilton syringe with a 30 gauge disposableneedle, the appropriate amount of virus was injected (10⁴-10⁶ PFU in 2μL) through a midline incision at the same stereotactic coordinates usedfor tumor cell injection. The injection was performed over 1 minute, andfollowing the injection the needle was left in place for 3 minutes, andthen slowly withdrawn.

Magnetic Resonance Imaging:

Mice were imaged using a 1.9 Tesla 30 cm bore animal MRI system locatedin the Hospital of the University of Pennsylvania MRI facility. Animalswere anesthetized with I.M. ketamine/xylazine (87 mg/kg ketamine/13mg/kg xylazine). Subsequently, each animal was injected with 10 units ofGd(DTPA) via a tail vein. The animal was taped in place within aplexiglass gradient coil and imaged.

Immunohistochemistry:

HSV-infected cells were detected by an indirect avidin-biotinimmunoperoxidase method (Vectastain ABC Kit, Vector Labs, Burlingam,Calif.) as specified by the manufacturer with slight modification.Briefly, tissue sections were deparaffinized, rehydrated, quenched inperoxide (H₂O₂) and blocked in 3.5% goat serum (Sigma Chem. Co., St.Louis, Mo.) Tissue sections were incubated overnight at 4° C. with theprimary antibody, a rabbit antiserum to HSV-1 (Dako Corp., Carpinteria,Calif.), used at a dilution of 1:1000. Next, the tissue was incubated atroom temperature with biotinylated goat anti-rabbit IgG, theavidin-biotin horseradish peroxidase complex and finally AEC substrate.Sections were counter stained with hematoxylin and examined under thelight microscope. As a control for the specificity of immuno-staining,tissues were processed as above, except that non-immune rabbit serum wassubstituted for the primary HSV-1 antiserum.

Titration of Virus from Tumor and Brain:

Mice were sacrificed by lethal injection anesthesia. Brains with orwithout in situ tumors were removed aseptically, snap frozen in liquidnitrogen, and stored at −70° C. Each tissue sample was rapidly thawed ina 37° C. water bath, and the tissue was homogenized in viral culturemedium at a 10% weight/volume ratio using a Pyrex Ten Broeck tissuegrinder. The homogenates were centrifuged at 3,000×g for 10 minutes at4° C. The supernatant of each tissue homogenate was dilutedlogarithmically in media, and the viral titer of each was determined byplaque assay on BHK cells (28).

Statistics:

Standard deviation, and t-Test: two sample assuming unequal variances,were calculated using Microsoft Excel (Redmond, Wash.) on an appleMacintosh computer (Cupertino, Calif.).

Example 1 Lysis of Melanoma Cells

In our initial studies, we wanted to make a straightforward in vitrodetermination of the relative abilities of HSV-1 wild type and mutantviruses to lyse various murine melanoma cells. We also wanted to comparehow efficiently these melanoma cells were lysed by HSV-1 relative tobaby hamster kidney (BHK) cells, which is a standard cell line used topropagate and titer HSV-1. Cells were plated in 24 well tissue cultureplates at a density of 5×10⁴ cells/well. The viruses were dilutedlogarithmically and cell monolayers were infected in triplicate. After72 hours of culture, the highest dilution of virus at which completedestruction of the monolayer still occurred, was recorded for eachvirus-cell combination. Data are expressed as the number of PFU ofvirus, obtained for each virus-cell combination.

As demonstrated in Table 1, the various mutant viruses lyse melanomacells and BHK with efficiencies similar to wild type 17+. Cloudman S-91,and H-P melanoma cells were lysed efficiently relative to BHK.

Example 2 Tumor Production

The capacity of each melanoma cell line to produce intracranial tumorswas then evaluated. For each cell line, 10 C57B1/6 mice were injectedstereotactically with 5×10⁴ cells in the right cerebral hemisphere. Micewere observed daily, and sacrificed when they appeared moribund, orafter 6 weeks if they remained asymptomatic. Each brain was fixed,sectioned, stained, and examined histologically for tumor. Both H-P andB-16 formed intracranial tumors in 10 of 10 C57B1/6 mice, while CloudmanS-91 only formed a tumor in 1 of 10 mice.

We decided to proceed with the H-P model, since these cells were bothsusceptible to lysis by the relevant HSV-1 mutants, and formed braintumors efficiently.

Subsequent experiments verified that stereotactic injection of H-P cellsinto the brain of C57B1/6 mice establishes tumors in 100% of theanimals. A technical advantage of this system is that the presence of abrain tumor can be verified by magnetic resonance imaging (MRI) prior totreatment, or simply by observation of a pigmented area on the skulloverlying the tumor site, generally by 5 days post cell injection. Thetumors progressed to a size that caused the mice to become moribund fromneurologic symptoms in approximately two weeks.

Example 3 Treatment of Brain Tumors with HSV-1 Mutant 1716

C57B1/6 mice were injected stereotactically in the right cerebralhemisphere with 5×10⁴ Harding-Passey melanoma cells. After 10 days (FIG.1 a) or 5 days (FIG. 1 b), 5×10⁵ PFU of HSV 1716 was injected at thesame stereotactic coordinates. The number of days elapsed betweeninjection of tumor cells and time mice became moribund is shown on the Xaxis. Control mice were injected with an equal volume of viral culturemedium at the appropriate time.

As shown in FIG. 1 a stereotactic injection of HSV-1 mutant 1716 intobrain tumors 10 days after establishment, resulted in a statisticallysignificant increase in the length of time elapsed until the mice becomemoribund (P(T<=t) one-tail: 1.016×10⁻⁴). However, no long term survivorswere obtained. When viral therapy was performed 5 days after tumorestablishment (FIG. 1 b), significant improvement in outcome was againseen in the treatment group (P(T<=t) one-tail: 7.707×10⁻³), and 2/10treated mice were cured. One long term survivor was sacrificed after day39 post viral infection. Microscopic examination of serial sections ofthe brain did not reveal any residual tumor (data not shown). The secondanimal is still alive and asymptomatic at greater than 150 days posttreatment. Treated animals that became moribund, showed progression oftheir brain tumors upon examination of tissue sections.

Example 4 1716 Replication in Tumor and Non-Tumor Cells

Immunohistochemistry shows that replication of 1716 is in factrestricted to tumor cells, and does not occur in surrounding brain. Asignificant number of melanoma cells within tumor were stained bypolyclonal antiserum to HSV-1 on days 3 and 6 past infection. Moreover,in tumor bearing mice treated with 1716, no HSV-1 antigen staining wasseen in brain tissue adjacent to tumor or in any other areas of brain inall sections examined. In addition, no histologic evidence ofencephalitis was seen in any 1716 treated mice at any time. In contrast,tumor bearing mice infected with wild type 17+ virus, exhibited multiplefocal areas of HSV-1 immunohistochemical staining both within tumor andin surrounding and distant brain as well. A significant encephalitischaracterised by polymorphonuclear leukocytes, nuclear dust, andextravasation of red blood cells, is seen in areas of this and othersections examined. In control experiments, no immunohistochemicalstaining was seen with anti-HSV-1 in tumor or brain from mice who didnot receive virus, or in virally infected brain tumor sections subjectedto the full immunohistochemical protocol with normal rabbit serumsubstituted for the primary anti-HSV-1 antibody (data not shown).

Example 5 Kinetics of Replication in Tumor and Non-Tumor Cells

Having shown striking restriction of 1716 replication to tumor byimmunohistochemistry, we next attempted to quantify the kinetics andextent of replication of 1716 in tumor by titration of infectious virus,and compare this with titration data from non-tumor bearing mouse braininfected with 1716 or 17+.

To investigate the extent of 1716 replication in brain tumors, C57B1/6mice were injected with Harding Passey melanoma cells right of midline.Seven days later each mouse was infected with 5×10⁵ PFU of 1716 at thesame stereotactic coordinates. At the times indicated, mice weresacrificed, the brains were frozen in liquid N₂ and stored at −70° C.Specimens were thawed rapidly, homogenized, and viral titration wasperformed in triplicate on BHK cells (closed squares). These datarepresent the mean of 4 mice at each time point.

To establish the growth characteristic of 1716 and wild type 17+ inbrain without tumor, mice were injected intracranially with either 5×10⁵PFU 1716 (open squares) or 1×10³ PFU of 17+ (closed triangles). Micewere sacrificed at the times shown and processed as described above.Each point is the mean of 2 mice.

As shown in FIG. 2, wild type 17+ virus replicated efficiently innon-tumor bearing mouse brain. In contrast, no replication of 1716occurred in brain of non-tumor bearing mice. The titer of virusrecovered decayed over time, and infectious 1716 could only be isolatedfor 3 days after inoculation. However, when 1716 was injected into braintumors, significant replication occurred as evidenced by recovery of anamount of infectious 1716 on day 1 post inoculation that issubstantially greater than the input amount. Under these conditions,infectious 1716 could be isolated from tumor bearing mice for 5 dayspost inoculation, but not on day 7. These results clearly demonstratethat HSV-1 mutant 1716 will freely replicate in tumor cells (leading totheir destruction) but does not replicate in non-tumor cells (leavingthem unharmed).

TABLE 1 Relative susceptibility of melanoma cells to lysis by HSV-1.Cell Type Cloudman Harding- Virus S91 Passey BHK dlspTK 10³ 10⁴ >10³1716 10⁴ 10⁴ −10³ 17+ (wild type) 10³ 10³ >10²

EXAMPLES Section 2 Materials & Methods Virus Stocks

To produce virus stocks, subconfluent monolayers of baby hamster kidney21 clone 13 (BHK) cells were infected with HSV strains 1716, in 1814, orparental 17⁺. Strain in 1814 has a mutation (insertion) in the VP16 gene(located in the U_(L) region; FIG. 3A) and strain 1716 has a mutation(deletion) in the γ34.5 gene (mutant; FIG. 3C). Virus was concentratedfrom the culture, titered on BHK cells by plaque assay and stored at−70° C. in 0.5 ml aliquots of viral culture medium (AUTO-POW mediacontaining penicillin and streptomycin) and thawed rapidly just prior touse as described (30, 34).

Culture of Tumor Cells and Differentiation of NT2

NTera-2 (clone D1) cells (referred to here as NT2 cells) were culturedas described (28, 29). Briefly, the cells were passaged 1:3 twice perweek in OptiMEM with 5% fetal bovine serum (FBS) andpenicillin/streptomycin (P/S). The medulloblastoma cell lines, D283 MEDand DAOY, were cultured in RPMI 1640 with 10% FBS, 1% P/S and 1%Glutamine. BHK cells were cultured in AUTO-POW with 5% FBS, 1% P/S and1% Glutamine. NT2 cells were plated at a density of 2.0×10⁶ in T75flask, and fed twice weekly with DMEM-HG supplemented with 10% FBS, 1%P/S, and 10⁻⁵M retinoic acid for 5 weeks. The differentiated NT2N cellswere separated from non-neuronal cells as described (29, 35). On the dayof intracranial injection, NT2 cells in sub-confluent monolayer culturewere harvested, washed three times in buffer and placed on a bed of iceuntil injected into the brains of nude mice.

Plague Assay

NT2, BHK, DAOY and D283 MED cells were plated in 24 well tissue cultureplates at a density of 10⁵ cells/well. The ciruses of interest werediluted logarithmically and cell monolayers were infected in triplicatewith multiplicity of infections (MOI) ranging from 10 to 0.01. Cultureswere observed regularly for the degree of cytopathic effects (CPE) ofthe viruses and noted for each MOI.

Titration of Virus from Cell Cultures

Cells were infected at MOI=1, harvested at 4, 24 and 48 hrspost-infection and stored at −70° C. The samples were freeze-thawedtwice from −70° C. to 37° C., centrifuged at 3,000×g for 10 minutes at40° C., the supernatant was diluted logarithmically in media and theviral titer of each sample was determined by plaque assay on BHK cells(34).

Titration of Virus from Tumor and Brain

To titrate viral inoculums in tumor and brain, nude mice wereintracranially inoculated with 1×10⁶ PFU of strain 17⁺ or 6.25×10⁴ PFUof strain 1716 and the mice were sacrificed by lethal injection ofanesthesia (ketamine/xylazine). The brains and tumors were dissectedfrom mice that were sacrificed on different days post viral inoculation(day 0, 1, 3, 5 and 7), quick frozen in liquid nitrogen and stored at−70° C. The brain and tumor samples from the different time points wererapidly thawed in a 37° C. water bath, and the tissue was homogenized inviral culture medium at a 10% weight/volume ratio using a Pyrex TenBroeck tissue grinder. The homogenates were centrifuged at 3,000×g for10 minutes at 4° C., the supernatant was diluted logarithmically inmedia and the viral titer of each sample was determined by plaque assayon BHK cells (34).

Intracerebral Graft Implantation

Female homozygous nude mice (4 to 6 weeks old) were obtained from HarlanSprague Dawley (Indianapolis, Ind.), the mice were anesthetized withintramuscular (IM) ketamine/xylazine (87 mg/kg ketamine/13 mg/kgxylazine) and stereotactic injections of tumor cell suspensions wereperformed using a small animal stereotactic apparatus (Kopf instruments,Tujunga, Calif.), a 10 μl Hamilton syringe and a 30 gauge disposableneedle as previously described (35). To make cortical tumors, thesyringe needle was positioned at a point 2 mm rostral of the bregma and1 mm to the right of midline. The skull was cleansed with 70% ethanoland perforated with a 27 gauge needle and the Hamilton syringe with theattached needle was advanced through the hole in the skull to a depth of1.5 mm below the dura and 3×10⁴ NT2 cells in a total volume of 2 μl wereinjected over 5 min. Prior to implantation, NT2N cells were suspended inDMEM/HG and maintained at 40° C. in an ice bath. Exactly 5 μl of theNT2N cell suspension, containing approximately 5×10⁵ cells, was injectedat the same location as above. Following the injection, the needle wasleft in place for 5 min and then slowly withdrawn over 2 min. and thesuperficial skin wound was closed with sutures. The mice were allowed torecover and inspected daily for signs of illness. Body weight andcranial measurements with calipers were taken weekly. Any mice thatshowed signs of morbidity in extremis were sacrificed and brains wereprepared for histochemistry. Tissues from some animals that diedunobserved in their cage also were harvested and fixed for histochemicalanalysis. The experiments on nude mice are summarised in Table 2.

Viral Inoculation

Control mice and mice previously inoculated with tumor cells wereanesthetized as described above and the head was cleansed with 70%ethanol. Using a Hamilton syringe with a 30 gauge disposable needle, theappropriate amount of virus was injected (10⁴-10⁶ PFU in 5 μl) through amidline incision at the same stereotactic coordinates used for theprevious injection of tumor cells. The injection was performed over 3min following the injection and then slowly withdrawn over 1 min.Control mice received equivalent volume inoculations of viral medium.

Magnetic Resonance Imaging

The brains of selected mice were imaged using a 30 cm bore 1.9 Teslaanimal Magnetic Resonance Imaging MRI) system (General Electric). Toaccomplish this, mice were anesthetized as described above at varioustimes after implantation of tumor cells and inoculation of the tumorsites with virus. Subsequently, each animal was injected with 10 unitsof an enhancing agent, gadolinium complexed to a DTPA carrier(Magnevist), via a tail vein. The animal was then immobilised within aPlexiglas RF coil and imaged.

Immunohistochemical Procedures

Mice were transcardially perfused and fixed with 70% ethanol in isotonicsaline (150 nM NaCl, pH 7.4) or 4% paraformaldehyde (0.1M PBS·pH 7.4)and the brain as well as samples of multiple other tissues (i.e.trigeminal ganglions, heart, proximal jejunum, liver, spleen, leftkidney, femur, and vertebral bodies) were dissected for histological andimmunohistochemical analysis. The methods for tissue processing andlight microscopic immunohistochemical analysis were similar to thosedescribed elsewhere (35, 36). Both monoclonal and polyclonal antibodiesto neuronal and glial cytoskeletal proteins and other polypeptides thathave been shown to serve as molecular signatures of the neuronal orglial phenotype were used for the immunohistochemical characterisationof intracranial allografts (35, 37). Rabbit polyclonal antisera to HSV-1which detects the major glycoproteins present in the viral envelope andat least one core protein (Dako corp., Carpinteria, Calif.) was used ata dilution of 1:1000 to detect replicating virus (38). A mousemonoclonal antibody (MOC-1) to neural cell adhesion molecule (NCAMs)specific for human NCAMs was used at a dilution of 1:100 to detect NT2and NT2N cells and to distinguish them from mouse brain cells (35).Another monoclonal antibody, RMO93 (1:10), which recognises rodentspecific epitopes of the middle molecular weight neurofilament (NF-M)protein and does not cross-react with human NF-M was used to confirm theidentity of NT2N grafts (35). RMO301 (1:100) is a monoclonal antibodythat recognises human specific NF-M was used to confirm NT2N grafts.M13, a mouse monoclonal antibody which recognises human microtubuleassociated protein-2 (MAP2), was used at a 1:500 dilution. Rabbitpolyclonal antibody specific to mouse myelin basic protein (MBP) wasused at a dilution of 1:1000 (gift of A. McMorris). Tissue sections forstaining with M1B-1 (a mouse monoclonal antibody that recognises acell-cycle specific antigen (Ki-67) used at a 1:20 dilution; AMAC,Westbrook, Me.) were pretreated by microwaving on 10 mM Sodium Citrateas described (39). Prior to sacrifice some control mice were injectedwith intraperitoneally with bromodeoxyuridine (BrdU) at 5 mg/g (in 150mM NaCl, 7 mM NaOH) body weight in order to label NT2 cells undergoingcell division in the grafts as described (37). Segments of the proximalintestine were removed from the same mouse as positive controls forcycling cells. BrdU positive cells were identified by using an BrdUantibody BU-33 (1:250). Antigen expressing cells were detected by theindirect avidin-biotin immunoperoxidase (Vectastain ABC kit, VectorLabs, Burlingam, Calif.) or peroxidase anti-peroxidase detection systemswith 3,3′-diaminobenzadine (DAB) as the chromagen. Grafts and spread ofvirus in all animals was monitored by screening every tenth sectionthrough entire brain with MOC-1, MIB-1 and HSV antibodies.

In Situ Hybridization for HSV-1 Specific Gene Expression.

Sections of perfused and fixed tissue were mounted on slides and in situhybridization performed as previously described to detect viral geneexpression (26, 33, 40). Serial tissue sections were hybridized with a³⁵S-labeled HSV LAT specific probe (BstEII-BstEII, FIG. 1E), with a³⁵S-labeled HSV specific thymidine kinase probe (tk; an early geneproduct) or with a biotinylated HSV specific gC (a late gene product)probe.

Preparation of ³⁵S-Labeled Nick-Translated Probe

The latency associated transcript (LAT) probe BstEII-BstEII subfragment(0.9 kb) of BamHI B was isolated from restriction digests by gelelectrophoresis and purified by GeneClean (Bio 101 Inc.; La Jolla,Calif.; see FIG. 3) (30). The 3.4 kb BamHI fragment encoding the tk genewas isolated as described (41). DNA probes were nick-translated andseparated from unicorporated nucleotides by passage through SephadexG-50 spin columns (Pharamacia) (33). The specific activities of theprobes were approximately 1-5×10⁸ c.p.m/μg DNA.

Preparation of ³⁵S-Labeled Poly (dT) Probe

A 21-mer of poly(dT) was synthesized and was used as substrate forlabeling by terminal deoxynucleotidyl transferase (TdT). Reaction mixconsisted of 2 μl of TdT, 1 μl of Poly(dT) (6 μg/μl), 5 μl of 5×TdTBuffer, 6 μl of CoCl₂ (2.5 mM), 10 μl of ³⁵S-dTTP (1 μg), 1 μl of ddH₂O.The mix was incubated at 37° C. for 30 min. and was separated fromunincorporated nucleotides by passage through Sephadex G-25 spincolumns. In situ hybridization was performed exactly as above exceptthat hybridization and washes were performed at 37° in 25% formamide(42). Exposure time courses were performed on uninfected, mock infected,wild-type virus infected and RNase treated tissue sections and were usedas positive and negative controls for experimental tissue sections (see43).

Biotinylated gC Probe to Detect Active Viral Replication.

A nonisotopic in situ hybridization was performed using a 21 bpantisense gC probe (nucleic acids 199-219 of gC transcript,CGGGGCGGGCGTGGCCGGGGG; gift by K. Montone; FIG. 3F) linked to the 3′ endby a biotinylated tail [5′-(TAG)₂-BBB-3′]. The protocol was essentiallythe same as in Wang and Montone with slight modifications for mousebrain tissue.

DNA Nick End Labeling by TUNEL Method.

The terminal deoxynucleotidyl transferase (TdT) dUTP-biotin nick endlabeling (TUNEL) technique was performed as previously described (37).Briefly, deparaffinized and rehydrated slides were digested with 20μg/ml of proteinase K in 0.1 M Tris (pH=8) at room temperature for 15min. After washing, the sections were incubated with a mixturecontaining 20 mM biotinylated-dUTP, 0.3 U/μl terminal deoxynucleotidyltransferase, 1.5 mM cobalt chloride, 200 mM sodium cacodylate, 25 mMTris, 0.25 mg/ml bovine serum albumin (pH=6.6) at 37° C. for 45 min. Thereactions were stopped by washing in 2×SSC for 15 min. and the resultswere visualized by alkaline phosphatase conjugated with streptavidin anddeveloped with Fast Red substrate. Coronal sections of post-natal dayeight rat brain were used as positive controls for this TUNEL protocolbecause this was the developmental stage at which peak apoptosisactivity was recognised (44).

Electron Microscopy of Thin Sections

Portions of tumor tissues from perfused mouse brains were fixed in 1%glutaraldehyde, 4% paraformaldehyde in 0.1M sodium cacodylate (pH=7.4)over night at 4° C. and washed in sodium cacodylate buffer and processedfor EM as described (37).

Statistics

Survival and weight statistics were performed using BMDP StatisticsSoftware (ed. W J Dixon; Release 7.0; 1993). Differences in survival incontrol and treated groups were compared using Generalised Wilcoxon(Breslow) Analysis. Differences in weights were compared using thet-test and the Mann-Whitney Test. Moribund animals in extremis weresacrificed and treated the same as animals found dead in their cage forstatistical analysis.

Example 6 HSV-1 Strain 1716 Lyses and Spreads on Monolayers of TumorDerived Human Neural Cell Lines In Vitro

To determine how efficiently HSV-1 strain 1716 lyses rapidly dividingNT2 in comparison with parental strain 17⁺, NT2 cells were plated on 24well plates 1 day prior to infection by these two strains atMultiplicities of Infection (MOI) of 10, 1 and to 0.1. Both viruses, atMOI of 10, lysed NT2 cells within 1 day and this was associated with thecharacteristic morphological changes (rounding up, phase brightness,cytomegaly, plaque formation and loss of adhesion) associated with HSVinfection. Since the behaviour of a virus at a low MOI (0.1) in vitromay predict the ability of a virus to spread in a tumor in vivo, westudied infection at MOI-0.1. Strain 1716 spread and destroyedmonolayers of NT2 cells less efficiently than 17⁺ (1716 lysed monolayerin 3 days and 17⁺ in 2 days). The behaviour of these viruses was similarin two different human medulloblastoma cell lines (D283 MED and DAOY)suggesting that strain 1716 can lyse many different brain tumor celllines.

Next, NT2N cells (the neuron-like retinoic acid differentiatedderivitatives of NT2 cells) were infected with these viruses. Strain1716 was attenuated for cytopathicity in these cells with respect tostrain 17⁺ and Lactate Dehydrogenase (LDH) assays for cytotoxicityperformed on NT2N cells infected with the above viruses showed that bothviruses caused some non-specific toxicity within 12 hours afterinfection (data not shown). Interestingly, titration of virus frominfected cell cultures showed that strain 1716 was deficient forreplication in NT2N cells (data not shown). Because strain 1716 is moreseverely neuroattenuated in mice than the other engineered strains (26,30), we conducted in vivo studies of strain 1716 versus 17⁺ virusinoculated into the CNS of nude mice with and without transplants of invitro derived NT2N cells or with tumors established from transplantedNT2 cells.

Reference is made to Table 2.

Example 7 Replication of HSV-1 Strain 1746 Cannot be Detected in theMouse CNS Following Intracerebral Inoculation

Consistent with previous results using SCID mice (26) intracerebral (IC)inoculation of 5×10⁶ plaque forming units (PFU) of strain 1716 in nudemice did not induce clinical symptoms for over 4 monthspost-inoculation, and there was no evidence of encephalitis onhistological analysis of the brains nor any evidence of replication inthe major organs (e.g. liver, spleen, bone marrow, etc.) of these mice(Table 2, Study I). In contrast to strain 1716, IC inoculation of lessthan 100 PFU of strain 17⁺ killed nude mice within 5-10 days andhistopathological analysis revealed extensive cytopathic lesions (e.g.intranuclear inclusion bodies, cell death, etc.) in these mice (notshown).

To monitor viral replication in the brain after IC inoculation of strain1716 versus strain 17⁺, a viral titration assay was performed (Table 2,Study II). The Recovery of both viruses on day 0 was low relative to theamount of virus in the injected inoculum. This probably was due toadsorption or fusion of the viral particles to the membranes in thebrain homogenates and to inactivation of virus during harvesting. FIG. 4shows that the titer of strain 17⁺ exponentially increased with time andresulted in morbidity and death of inoculated mice. In contrast, thetiter of strain 1716 dropped precipitously in the brains of nude mice,and virus was no longer detectable 3 days post-inoculation. Moreover,there was no immunohistochemical evidence of encephalitis in strain 1716infected mice, and there was no detectable spread of strain 1716 virusoutside the CNS as evidenced by the absence of virus in samples ofliver, spleen, kidney, jejunum and bone marrow by immunohistochemistryand by in situ hybridization for HSV specific transcripts (data notshown). Likewise, direct inoculation of liver or intravenous injectionwith strain 1716 did not cause any morbidity or death in nude mice. Incontrast to strain 1716, strain 17⁺ infected mice exhibited evidence ofencephalitis and tumor lysis (see FIG. 4).

Example 8 HSV-1 Strain 1716 Lytically Replicates in NT2 Tumors but no inTransplanted NT2N Cells in the Mouse CNS

When strain 1716 was injected into NT2 tumors significant replicationoccurred for 7 days as evidenced by increase in viral titer over inputinoculum by day 3 (FIG. 4). This is in agreement with theimmunohistochemical and in situ hybridization data which showed nodetectable strain 1716 in the brains of these mice except in the NT2tumor cells for as long as they were present to support viralreplication (see below).

Since quantification by titration assay in mice showed that strain 1716replicated in NT2 cell tumors, we tested the ability of strain 1716 toinduce regression of these tumors (Table 2, Study IV). To do this weinjected 5 μl of strain 1716 containing 5×10⁵ PFU into tumors thatformed in the brains of nude mice following IC implantation of 3×10⁶ NT2cells. To monitor the fate of transplanted NT2 cells, mice weresacrificed at different time points after the viral inoculation andtheir brains and organs were analysed by immunohistochemistry and insitu hybridization. NT2 cells formed tumors with a neural and epithelialhistology in 100% of mice, and these tumors were lethal within 5 weeksafter grafting (FIG. 5A, 5B). These tumors contained abundantproliferating cells as evidenced by BrdU labeling, theimmunohistochemical detection of cell cycle antigens and their rapidgrowth (FIG. 5C, 5D). FIGS. 5E,F,G and H show that infection of NT2tumors with strain 1716 is not uniform at day 3 post-infection (pi).This may reflect the localised nature of the injection site, or tocell-type specific differences in the vulnerability of the cells toinfection and lysis by strain 1716. Nonetheless, most tumor cell typesnear the injection site harbored immunoreactive virus by day 5. At laterdays post-infection, more cells were infected, but immunoreactivity forvirus in the tumor was weaker presumably due to clearance of the virusfollowing lysis of the infected tumor cells (FIG. 5).

As seen in FIG. 5H, viral antigen is limited to the tumors. On highmagnification, the infected tumor cells showed characteristic featuresof HSV-1 infection, i.e. intranuclear inclusion bodies and multinucleargiant cell formation (FIG. 5F). No viral antigen staining was seen inthe surrounding brains of these mice or in the brains of controluntreated mice. This was confirmed by a non-isotopic in situhybridization protocol using a biotinylated probe for glycoprotein C(gC) and a radiolabeled thymidine kinase (TK) probe (a late and earlygene product expressed only in acute infection) to detect active viralreplication in serial sections (FIG. 5J). Thus, viral replication, asevidenced by gene expression, is also restricted to the tumor cells. Incontrast, tumor-bearing mice inoculated with strain 17⁺ showed viralreplication in both tumor and brain (FIG. 5K, 5L). Mouse brainsharvested 18 days after viral treatment of their tumor implants with1716 showed marked decrease in the size of tumor. Indeed only a residualfibrotic scar was seen in some mice, and viral antigen was strictlylimited to the remaining cells in the scar (FIG. 5 M, N, O).

To examine the ability of strain 1716 to induce regression of braintumors, NT2 tumors in the brains of nude mice were stereotacticallyinjected with strain 1716 twelve days after implantation of NT2 cells.Treated mice were inoculated with 5×10⁵ PFU (in 5 μl) of strain 1716 atthe tumor implantation site and control mice received 5 μl medium alone.MRI scans showed that these mice developed detectable tumors by 11 dayspost-implantation. In all mock treated tumor-bearing mice, the tumorgrew rapidly in size and was lethan (FIG. 6A-D). In all treated mice,strain 1716 infection induced a detectable regression of the tumor atthe original inoculation site (FIG. 6E-G).

To determine whether transplanted NT2N cells were permissive for HSVreplication, 2.5×10⁵ cells were transplanted into the brain parenchymaor ventricles of nude mice (Table 1; Study III). These cells integrateand survive for over a year and acquire a fully mature post-natal humanCNS neuronal phenotype (27). Strain 1716 was then inoculated at the samestereotactic site 6 weeks post-implantation. The grafts were identifiedand distinguished from mouse cells by using human specific (MOC1 andRMO301) and mouse specific (RMO93) antibodies to neural cell adhesionmolecule (MOC1) or neurofilament proteins (RMO301, RMO93). In contrastto NT2 tumors, these long-term NT2N transplants were non-permissive forstrain 1716 replication as evidenced by lack of immunohistochemicalstaining for viral antigens at days 1, 3, 5, 7, 9, 21 and 50 post-viralinoculation.

Example 9 HSV Strain 1716 Induces a Non-Apoptotic Death in NT2 TumorCells

Sections of tumor from a selected group of strain 1716 infected mice anduninfected tumor controls were prepared for EM analysis to characterisethe mode of cell death in the NT2 cell tumors. The infected cells on H&Estaining had the characteristics typical of HSV infected cells (FIG.5F). Viral assembly domains can be seen in the nucleus of infectedcells. There was no evidence for an apoptotic mechanism of cell death inthe virally infected tumor cells by EM (FIG. 7). Tumor cells with viralparticles showed the fragmentation and dissolution of nuclei andorganelles as well as condensed and marginated DNA. Terminaldeoxynucleotidyl transferase (TdT) dUTP-biotin nick end labeling (TUNEL)and DNA gel electrophoresis studies did not show evidence of DNAfragmentation indicative of apoptosis (data not shown). Taken together,these findings indicate that strain 1716 induces the characteristiclytic infection in the NT2 cells in vitro and in vivo.

Example 10 Long-Term Survival of HSV Strain 1716 Treated Tumor-BearingMice

Based on the results of the studies described above, we analysed thesurvival of a cohort of tumor-bearing mice that were or were not treatedwith virus (Table 2, Studies V and VI). This also enabled us to assessthe long-term consequences of treatment with strain 1716 (see below).Twenty mice were inoculated with 3×10⁶ NT2 cells and several days latermice were split into two groups. Control mice received culture mediumand treated mice received 5×10⁶ PFU of strain 1716. In the firstsurvival experiment (Table 2, Study V) virus treatment was given at 12days post-tumor cell inoculation. There was only 1 (10%) long-termsurvivor in this group and there was no significant difference insurvival between control (Study VA) and treated (Study VB) animals(p=0.63, FIG. 8A). Histological examination of some control and treatedanimals showed that virus was replicating in the tumor. In the treatedanimals it appears that the tumor had already grown and spread to such asize that one virus treatment was not sufficient to induce regression ofthe tumor (data not shown). When virus treatment was given at 7 dayspost-tumor cell inoculation, 44% of the mice survived long-term and 100%of control mice died within 10 weeks (Table 2, Study VI; FIG. 6B). Thisresulted in a statistically significant improvement in survival (p<0.03;median survival time (controls)=44.75+/−5.24: median survival time(treated)=101.78+/−22.69).

In the survival experiments, significant weight loss was observed in asub-population of both mock-treated tumor-bearing mice (Table 2, StudyVIA) and strain 1716 treated tumor-bearing mice (Table 2, Study VIB).Since there was no difference in the average weights of these two groups(FIG. 8C) we separated the treated group (Study VIB) into two sub-groups(High-Responders (HR)—the long-term survivors (4 mice) andLow-Responders (LR)—mice that died (5 mice), FIG. 8C). We found asignificant difference in weight loss at 6 weeks (p<0.01) between groupsHR and LR and between groups LR and 1716 treated mice (Study 1) usingthe standard t-test (FIG. 8D). These data confirmed earlier observationsof weight loss in Study V, and they suggest that the weight loss may bedue to the toxic physiological effects of tumor growth, regression orlysis and not directly due to an effect of the viral infection of thebrain. Notably, some of the treated mice that died showed leakage oftumor cells from implantation site into ventricles and leptomeninges.Since this would lead to obstruction of the flow of cerebrospinal fluid,it is not surprising that some of these treated mice had survivalkinetics similar to control mice (FIG. 8A). Finally, intracranial volumeof the control mice with brain tumors increased by over 25% (indicatingtumor growth) while in treated mice intracranial volume did not increasesignificantly (see FIG. 6). The long-term survivors also did not have asignificant increase in intracranial volume (data not shown). Theseinterpretations are supported by the fact that mice inoculated withstrain 1716 alone did not show any clinical or histologicalcharacteristics of encephalitis at any time during the study

Long-term survivors from studies V and VI (Table 2) had no clinicalsymptoms, no atypical increase in intracranial volume, and no weightloss (FIG. 8C). These mice and mice inoculated with strain 1716 alonewere sacrificed and analysed for pathology and viral replication byimmunohistochemistry and in situ hybridisation. In the survivors, therewas only evidence of fibrotic scar tissue and dystrophic calcificationsbut no evidence of residual, live tumor cells (FIG. 9A,B,C).Immunohistochemical staining for cell cycle antigens (e.g. using MIB-1)was also negative suggesting the absence of any cycling NT2 cells in thebrain. Further, the brains of these mice were negative for herpesantigens indicating the absence of replicating virus, although in somemice in situ hybridization revealed the presence of latent HSV in thehippocampus (FIG. 9D). Surprisingly, latent virus was also found in thehippocampus of mice infected with strain 1716 along. However,examination of representative rostral to caudal levels of the brains ofall survivors using antibodies specific to HSV and to the human NT2cells (MOC-1, RMO301), did not reveal any evidence of active viralreplication nor any residual live tumor cells. To exclude the possibleoccurrence of other potential toxic sequelae of HSV-1 strains such asdemyelination, we probed sections from the brains of mice usingantibodies to myelin basic protein. Examination of mice injected withstrain 1716 alone as well as long-term survivors revealed no evidence ofdemyelination (FIG. 9E). Finally, we monitored the levels of poly(A)⁺RNAby in situ hybridisation using a radiolabeled poly(dT) probe to assessthe overall metabolic health of neurons (31, 32) and we found noquantifiable difference in the level of poly (A)⁺RNA between theLAT-positive cells of the long-term survivors versus the contralateralnon-LAT positive cells in the same mice and in uninfected, control mice(FIG. 9F).

TABLE 2 Summary of Animal Experiments Treatment Study Tumor Virus (p.i.^(a)) Number Survival Clinical Disease I. — 1716 4 100% (>16 wks) n.a.II.A —   17^(↑) 10   n.a. ^(a) Encephalitis B — 1716 10 n.a. n.a. C NT21716 (12) 10 n.a. n.a. III. NT2N 1716 (6 wks) 10 n.a. None IV.A NT2 — 10^(b) None B NT2 1716 (14) 10 ^(b) None C NT2  17^(↓) (14) 2 ^(b)Encephalitis V.A NT2 — 10 0% (<8 wks) Cachexia ^(c) B NT2 1716 (12) 1010% (>35 wks) ^(e) None ^(d) VI.A NT2 — 8 0% (<10 wks) Cachexia ^(c) BNT2 1716 (7) 9 44% (>25 wks) ^(f) None ^(d) ^(a) p.i.—post cellimplantation when virus or mock inoculum was administered. n.a.—notapplicable. ^(b) mice were sacrificed at different days after viralinoculation to follow the kinetics of tumor progression and viral spread(see text for details). ^(c) These mice also showed no symptoms but themice that died showed the same range of symptoms as in mock treatedmice. ^(d) Long-term survivors showed no symptoms but the mice that diedshowed same range of symptoms as in mock treated mice. ^(e) p = 0.63, nostatistically significant difference in survival between mock andtreated mice. ^(f) p < 0.03, statistically significant difference insurvival between mock and treated mice.

CONCLUSION

HSV type I (HSV-1) strain 1716 has a deletion in the γ34.5neurovirulence gene which renders it avirulent in the mouse CNS, we haveassessed its potential to induce selective lysis of tumor cells versusneurons in vitro and in vivo. Parental HSV-1 strain 17⁺ and engineeredstrain 1716 were studied using human teratocarcinoma derived embryonalcarcinoma cells (NT2 cells). These cells resemble neuronal progenitorcells and can be induced to differentiate into neurons (NT2N) cells)with retinoic acid. Intracerebral grafts of NT2 cells into the brains ofnude mice resulted in lethal brain tumors while grafts of NT2N cellsresulted in the integration and maturation of NT2N cells withoutneoplastic reversion. In vitro studies showed that strain 1716replicates in and spreads on monolayers of NT2 cells, resulting in thelysis of these cells. However, strain 1716 did not replicate in NT2Ncells in vitro. In vivo, strain 1716 replicated preferentially in NT2tumors as evidenced by immunohistochemical staining for viral antigens,in situ hybridisation for HSV specific transcripts and by titration ofvirus from brains with tumor following intracranial injection of thevirus into these mice. In contrast to NT2 tumor cells, transplanted NT2Ncells were non-permissive for strain 1716 replication. The temporalregression of NT2 tumors in mice treated with strain 1716 wasdemonstrated in vivo by Magnetic Resonance Imaging. Electron microscopyand studies of DNA fragmentation suggested that regression of NT2 braintumors in strain 1716 treated mice was mainly due to a non-apoptotic,lytic mode of cell death. Strain 1716 treated NT2 tumor-bearing micesurvived over twice as long as mock-treated tumor bearing mice and thesedifferences in survival (25 vs. 9 wks.) were statistically significant(p<0.03). We conclude from these studies that strain 1716, areplication-competent, non-neurovirulent mutant of HSV-1, inducesregression of human neural tumors established in the brains of nude miceresulting in their prolonged survival. These results indicate that HSV-1γ34.5 mutants are candidates for the treatment of human brain tumors invivo.

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1. A method of treating a melanoma in a human, the method comprising thestep of administering herpes simplex virus type 1 (HSV-1) to said human,wherein the HSV-1 comprises a mutation in each γ34.5 gene, wherein theHSV-1 is avirulent, and wherein the HSV-1 infects, replicates within,and lyses tumor cells of the melanoma.
 2. A method according to claim 1wherein the step of administering the HSV-1 comprises intratumoralinjection of the HSV-1.
 3. A method according to claim 1 wherein thestep of administering the HSV-1 comprises injection of the HSV-1 intothe blood stream feeding the melanoma.
 4. A method according to claim 1wherein the HSV-1 is a strain 17 virus.
 5. A method according to claim 1wherein said mutation is within the BamHI s restriction fragment of theR_(L) terminal repeat corresponding to between 0-0.02 and 0.81-0.83 mapunits (mu).
 6. A method according to claim 1 wherein said mutationcomprises a deletion within the BamHI s restriction fragment of theR_(L) terminal repeat corresponding to between 0-0.02 and 0.81-0.83 mapunits (mu), wherein the deletion is from 0.1 to 3 kb.
 7. A methodaccording to claim 1 wherein said mutation comprises a deletion withinthe BamHI s restriction fragment of the R_(L) terminal repeatcorresponding to between 0-0.02 and 0.81-0.83 map units (mu), whereinthe deletion is from 0.7 to 2.5 kb.
 8. A method according to claim 1wherein said mutation comprises a deletion within the BamHI srestriction fragment of the R_(L) terminal repeat corresponding tobetween 0-0.02 and 0.81-0.83 map units (mu), wherein the deletion is a759 bp deletion in the 34.5 gene.
 9. A method according to claim 1wherein the mutation is a deletion, insertion, or substitution.
 10. Amethod according to claim 1 wherein the genome of said HSV-1 consists ofthe genome of a wild type HSV-1 virus having a mutation in each γ34.5gene.
 11. A method according to claim 1 wherein the HSV-1 is HSV1716.12. A method of treating a melanoma in a human, the method comprisingthe step of administering HSV1716 to said human, wherein the HSV1716 isavirulent, and wherein the HSV1716 infects, replicates within, and lysestumor cells of the melanoma.
 13. A method according to claim 12 whereinthe step of administering the HSV1716 comprises intratumoral injectionof HSV1716.
 14. A method according to claim 12 wherein the step ofadministering the HSV1716 comprises injection into the blood streamfeeding the tumor.
 15. The method of claim 1, wherein the HSV-1 ismutated, the mutation consisting of a mutation in each γ34.5 gene.